Vectors for Genetically-Encoded Tags for Electron Microscopy Contrast in DrosophilaReportar como inadecuado




Vectors for Genetically-Encoded Tags for Electron Microscopy Contrast in Drosophila - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Biological Procedures Online

, 18:5

First Online: 01 February 2016Received: 07 July 2015Accepted: 25 January 2016DOI: 10.1186-s12575-016-0034-1

Cite this article as: Tsui, M.M.K., Itoh, A., Amgad, M. et al. Biol Proced Online 2016 18: 5. doi:10.1186-s12575-016-0034-1

Abstract

BackgroundOne of the most notable recent advances in electron microscopy EM was the development of genetically-encoded EM tags, including the fluorescent flavoprotein Mini-SOG Mini-Singlet Oxygen Generator. Mini-SOG generates good EM contrast, thus providing a viable alternative to technically-demanding methods such as immuno-electron microcopy immuno-EM. Based on the Mini-SOG technology, in this paper, we describe the construction, validation and optimization of a series of vectors which allow expression of Mini-SOG in the Drosophila melanogaster genetic model system.

FindingsWe constructed a Mini-SOG tag that has been codon-optimized for expression in Drosophila DMS tag using PCR-mediated gene assembly. The photo-oxidation reaction triggered by DMS was then tested using these vectors in Drosophila cell lines. DMS tag did not affect the subcellular localization of the proteins we tested. More importantly, we demonstrated the utility of the DMS tag for EM in Drosophila by showing that it can produce robust photo-oxidation reactions in the presence of blue light and the substrate DAB; the resultant electron micrographs contain electron-dense regions corresponding to the protein of interest. The vectors we generated allow protein tagging at both termini, for constitutive and inducible protein expression, as well as the generation of transgenic lines by P-element transformation.

ConclusionsWe demonstrated the feasibility of Mini-SOG tagging in Drosophila. The constructed vectors will no doubt be a useful molecular tool for genetic tagging to facilitate high-resolution localization of proteins in Drosophila by electron microscopy.

KeywordsElectron microscopy Imaging Drosophila Mini-SOG Electronic supplementary materialThe online version of this article doi:10.1186-s12575-016-0034-1 contains supplementary material, which is available to authorized users.

Download fulltext PDF



Autor: Marco Man Kin Tsui - Anri Itoh - Mohamed Amgad - Shao-Fang Wang - Toshio Sasaki

Fuente: https://link.springer.com/







Documentos relacionados